Macular degeneration is a clinical term that is used to describe a variety of diseases that are all characterized by a progressive loss of central vision associated with abnormalities of Bruch""s membrane and the retinal pigment epithelium. These disorders include very common conditions that affect older patients (age related macular degeneration or AMD) as well as rarer, earlier-onset dystrophies that in some cases can be detected in the first decade of life1-18. The genes associated with some of these dystrophies have been mapped,5-14 and in four cases, blue-cone monochromasy,15 pattern dystrophy,16,17 and Sorsby fundus dystrophy,18 and Best Disease actually identified. However, none of the latter genes has been found to be responsible for a significant fraction of typical late-onset macular degeneration.
In developed countries, AMD is the most common cause of legal blindness in older patients.19 The hallmark of this condition is the presence of drusen, which are ophthalmoscopically visible, yellow-white hyaline excrescences of Bruch""s membrane. In some families, drusen are heritable in an autosomal dominant fashion.
In 1875, Hutchinson and Tay published a paper entitled xe2x80x9cSymmetrical Central Choroido-Retinal Disease Occurring in Senile Personsxe2x80x9d.20 This paper includes one of the first descriptions of the constellation of clinical findings now known as age related macular degeneration (AMD). Specifically, three of the ten patients in the report were sisters affected with whitish spots (now referred to as drusen) in the macula. In 1899, Doyne21 reported a similar disorder in which the abnormal spots were nearly confluent such that the macula had a xe2x80x9choneycombxe2x80x9d appearance. Histopathologic examination of one of Doyne""s patients22 revealed the abnormalities to be hyaline thickenings of Bruch""s membrane. In 1925, Vogt23 published the first description of the ophthalmoscopic appearance of a form of familial drusen that had been observed in patients living in the Leventine valley in the Ticino canton of southern Switzerland. Klainguti24 fully characterized this condition in 1932 and demonstrated its autosomal dominant inheritance. This disorder eventually became known as malattia leventinese, xe2x80x9cMLxe2x80x9d (i.e., Leventine disease). In 1948, Waardenburg25 stated that there was little reason to make a distinction between malattia leventinese and the condition described by Doyne, referred to as Doyne""s Honeycomb Retinal Dystrophy (DHRD). This position was strengthened when Forni and Babel26 found that the histopathologic features of malattia leventinese were indistinguishable from those of Doyne""s honeycomb choroiditis. Piguet, Haimovici and Bird27 recently reviewed the history of these conditions and also pointed out that the drusen in families with malattia leventinese are frequently distributed in a radical pattern. Choroidal neovascularization is uncommon in patients with radial drusen but does occur.27 Although originally recognized in Switzerland, families affected with autosomal dominant radial drusen have been identified in Czechoslovakia,28,29 and the United States.30 
In 1996, ML was mapped to chromosome 2p16-2148. Shortly thereafter, DHRD was mapped to the same locus49 and the genetic interval was narrowed48,49. ML and DHRD are very similar phenotypically to AMD.
Currently, there is no therapy that is capable of significantly slowing the degenerative progression of macular degeneration, and treatment is limited to laser photocoagulation of the subretinal neovascular membranes that occur in 10-15% of affected patients.
The present invention is based, at least in part, on the discovery of a novel human gene encoding a novel human protein, which has sequence homologies with fibulin (1 and 2), fibrillin, nidogen, notch, protein S and Factor IX. The newly identified proteins and nucleic acids described herein are referred to as xe2x80x9cEFEMPsxe2x80x9d. The human EFEMP1 gene (herein referred to as hEFEMP1) transcript is shown in FIG. 5 and includes 5xe2x80x2 and 3xe2x80x2 untranslated regions and a 1479 base pair open reading frame encoding a 493 amino acid polypeptide having SEQ ID NO. 1. Mouse EFEMP1 is expressed in eye, brain, heart, lung and kidney tissue.
In one aspect, the invention features isolated EFEMP1 nucleic acid molecules. In one embodiment, the EFEMP1 nucleic acid is from a vertebrate. In a preferred embodiment, the EFEMP1 nucleic acid is from a mammal, e.g. a human. In an even more preferred embodiment, the nucleic acid has the nucleic acid sequence set forth in FIG. 5 or a portion thereof. The disclosed molecules can be non-coding, (e.g. a probe, antisense, or ribozyme molecule) or can encode a functional EFEMP1 polypeptide (e.g. a polypeptide which specifically modulates biological activity, by acting as either an agonist or antagonist of at least one bioactivity of the human EFEMP1 polypeptide). In another embodiment, the nucleic acid of the present invention can hybridize to a vertebrate EFEMP1 gene or to the complement of a vertebrate EFEMP1 gene. In a further embodiment, the claimed nucleic acid can hybridize with a nucleic acid sequence shown in FIG. 5 or a complement thereof. In a preferred embodiment, the hybridization is conducted under mildly stringent or stringent conditions.
In further embodiments, the nucleic acid molecule is an EFEMP1 nucleic acid that is at least about 70%, preferably about 80%, more preferably about 85%, and even more preferably at least about 90% or 95% homologous to the nucleic acid shown as SEQ ID NO: 1 or to the complement of the nucleic acid shown as FIG. 5.
The invention also provides probes and primers comprising substantially purified oligonucleotides, which correspond to a region of nucleotide sequence which hybridizes to at least about 6, at least about 10, at least about 15, at least about 20, or preferably at least about 25 consecutive nucleotides of the sequence set forth as FIG. 5 or complements of the sequence set forth as FIG. 5 or naturally occurring mutants or allelic variants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto, which is capable of being detected.
For expression, the subject nucleic acids can be operably linked to a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter (e.g., for constitutive expression or inducible expression) or transcriptional enhancer sequence. Such regulatory sequences in conjunction with an EFEMP1 nucleic acid molecule can provide a useful vector for gene expression. This invention also describes host cells transfected with said expression vector whether prokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo (e.g. transgenic) methods for producing EFEMP 1 proteins by employing said expression vectors.
In another aspect, the invention features isolated EFEMP1 polypeptides, preferably substantially pure preparations, e.g. of plasma purified or recombinantly produced polypeptides. The EFEMP1 polypeptide can comprise a full length protein or can comprise smaller fragments corresponding to one or more particular motifs/domains, or fragments comprising at least about 6, 10, 25, 50, 75, 100, 125, 150, 200, 225, 250, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470 480 or 490 amino acids in length. In particularly preferred embodiments, the subject polypeptide has an EFEMP1 bioactivity.
In a preferred embodiment, the polypeptide is encoded by a nucleic acid which hybridizes with the nucleic acid sequence represented in FIG. 5. In a further preferred embodiment, the EFEMP1 polypeptide is comprised of the amino acid sequence set forth in SEQ ID NO. 1. The subject EFEMP1 protein also includes within its scope modified proteins, e.g. proteins which are resistant to post-translational modification, for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with intracellular proteins involved in signal transduction.
The EFEMP1 polypeptides of the present invention can be glycosylated, or conversely, by choice of the expression system or by modification of the protein sequence to preclude glycosylation, reduced carbohydrate analogs can also be provided. Glycosylated forms can be obtained based on derivatization with glycosaminoglycan chains. Also, EFEMP1 polypeptides can be generated which lack an endogenous signal sequence (though this is typically cleaved off even if present in the pro-form of the protein).
In yet another preferred embodiment, the invention features a purified or recombinant polypeptide, which has the ability to modulate, e.g., mimic or antagonize, an activity of a wild-type EFEMP1 protein. Preferably, the polypeptide comprises an amino acid sequence identical or homologous to a sequence designated in SEQ ID NO. 1.
Another aspect of the invention features chimeric molecules (e.g., fusion proteins) comprising an EFEMP1 protein. For instance, the EFEMP1 protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the EFEMP 1 polypeptide. A preferred EFEMP1 fusion protein is an immunoglobulin-EFEMP1 fusion protein, in which an immunoglobulin constant region is fused to an EFEMP1 polypeptide.
Yet another aspect of the present invention concerns an immunogen comprising an EFEMP1 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for an EFEMP1 polypeptide; e.g. a humoral response, an antibody response and/or cellular response. In a preferred embodiment, the immunogen comprises an antigenic determinant, e.g. a unique determinant of a protein encoded by the nucleic acid set forth in SEQ ID NO. 1 or as set forth in SEQ ID NO. 1.
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of an EFEMP1 protein.
The invention also features transgenic non-human animals which include (and preferably express) a heterologous form of an EFEMP1 gene described herein, or which misexpress an endogenous EFEMP1 gene (e.g., an animal in which expression of one or more of the subject MFGF proteins is disrupted). Such transgenic animals can serve as animal models for studying cellular and/or tissue disorders comprising mutated or mis-expressed EFEMP1 alleles or for use in drug screening. Alternatively, such transgenic animals can be useful for expressing recombinant EFEMP1 polypeptides.
The invention further features assays and kits for determining whether an individual""s EFEMP1 genes and/or proteins are defective or deficient (e.g in activity and/or level), and/or for determining the identity of EFEMP1 alleles. In one embodiment, the method comprises the step of determining the level of EFEMP1 protein, the level of EFEMP1 mRNA and/or the transcription rate of an EFEMP1 gene. In another preferred embodiment, the method comprises detecting, in a tissue of the subject, the presence or absence of a genetic alteration, which is characterized by at least one of the following: a deletion of one or more nucleotides from a gene; an addition of one or more nucleotides to the gene; a substitution of one or more nucleotides of the gene; a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; and/or a non-wild type level of the EFEMP1 protein. For example, detecting a genetic alteration or the presence of a specific polymorphic region can include (i) providing a probe/primer comprised of an oligonucleotide which hybridizes to a sense or antisense sequence of an EFEMP1 gene or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the EFEMP1 gene; (ii) contacting the probe/primer with an appropriate nucleic acid containing sample; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic alteration. Particularly preferred embodiments comprise: 1) sequencing at least a portion of an EFEMP1 gene, 2) performing a single strand conformation polymorphism (SSCP) analysis to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids; and 3) detecting or quantitating the level of an EFEMP1 protein in an immunoassay using an antibody which is specifically immunoreactive with a wild-type or mutated EFEMP1 protein.
Information obtained using the diagnostic assays described herein (alone or in conjunction with information on another genetic defect, which contributes to the same disease) is useful for diagnosing or confirming that a symptomatic subject has a genetic defect (e.g. in an EFEMP1 gene or in a gene that regulates the expression of an EFEMP1 gene), which causes or contributes to the particular disease or disorder. Alternatively, the information (alone or in conjunction with information on another genetic defect, which contributes to the same disease) can be used prognostically for predicting whether a non-symptomatic subject is likely to develop a disease or condition, which is caused by or contributed to by an abnormal EFEMP1 activity or protein level in a subject (e.g. a macular degeneration). In particular, the assays permit one to ascertain an individual""s predilection to develop a condition associated with a mutation in EFEMP1, where the mutation is a single nucleotide polymorphism (SNP). Based on the prognostic information, a doctor can recommend a regimen (e.g. diet or exercise) or therapeutic protocol useful for preventing or prolonging onset of the particular disease or condition in the individual.
In addition, knowledge of the particular alteration or alterations, resulting in defective or deficient EFEMP1 genes or proteins in an individual, alone or in conjunction with information on other genetic defects contributing to the same disease (the genetic profile of the particular disease) allows customization of therapy for a particular disease to the individual""s genetic profile, the goal of pharmacogenomics. For example, an individual""s EFEMP1 genetic profile or the genetic profile of a disease or condition to which EFEMP1 genetic alterations cause or contribute, can enable a doctor to: 1) more effectively prescribe a drug that will address the molecular basis of the disease or condition; and 2) better determine the appropriate dosage of a particular drug. For example, the expression level of EFEMP1 proteins, alone or in conjunction with the expression level of other genes known to contribute to the same disease, can be measured in many patients at various stages of the disease to generate a transcriptional or expression profile of the disease. Expression patterns of individual patients can then be compared to the expression profile of the disease to determine the appropriate drug and dose to administer to the patient.
The ability to target populations expected to show the highest clinical benefit, based on the EFEMP1 or disease genetic profile, can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup-specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labeling (e.g. since the use of EFEMP1 as a marker is useful for optimizing effective dose).
In another aspect, the invention provides methods for identifying a compound which modulates an EFEMP1 activity, e.g. the interaction between an EFEMP1 polypeptide and a target peptide. In a preferred embodiment, the method includes the steps of (a) forming a reaction mixture including: (i) an EFEMP1 polypeptide, (ii) an EFEMP1 binding partner, and (iii) a test compound; and (b) detecting interaction of the EFEMP1 polypeptide and the EFEMP1 binding protein. A statistically significant change (potentiation or inhibition) in the interaction of the EFEMP1 polypeptide and EFEMP1 binding protein in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of EFEMP 1 bioactivity for the test compound. The reaction mixture can be a cell-free protein preparation, e.g., a reconstituted protein mixture or a cell lysate, or it can be a recombinant cell including a heterologous nucleic acid recombinantly expressing the EFEMP1 binding partner.
In preferred embodiments, the step of detecting interaction of the EFEMP1 and EFEMP1 binding partner is a competitive binding assay. In other preferred embodiments, at least one of the EFEMP1 polypeptide and the EFEMP1 binding partner comprises a detectable label, and interaction of the EFEMP1 and EFEMP1 binding partner is quantified by detecting the label in the complex. The detectable label can be, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. In other embodiments, the complex is detected by an immunoassay.
Yet another exemplary embodiment provides an assay for screening test compounds to identify agents which modulate the amount of EFEMP1 produced by a cell. In one embodiment, the screening assay comprises contacting a cell transfected with a reporter gene operably linked to an EFEMP1 promoter with a test compound and determining the level of expression of the reporter gene. The reporter gene can encode, e.g., a gene product that gives rise to a detectable signal such as: color, fluorescence, luminescence, cell viability, relief of a cell nutritional requirement, cell growth, and drug resistance. For example, the reporter gene can encode a gene product selected from the group consisting of chloramphenicol acetyl transferase, luciferase, beta-galactosidase and alkaline phosphatase.
Also within the scope of the invention are methods for treating diseases or disorders which are associated with an aberrant EFEMP1 level or activity or which can benefit from modulation of the activity or level of EFEMP1 (e.g. a macular degeneration). The methods comprise administering, e.g., either locally or systemically to a subject, a pharmaceutically effective amount of a composition comprising an EFEMP1 therapeutic (e.g. an MD therapeutic).
Other features and advantages of the invention will be apparent from the following detailed description and claims.